Distal Substrate Interactions Enhance Plasmepsin Activity

Istvan, Eva S.; Goldberg, Daniel E.

doi:10.1074/jbc.m412086200

Cited by 27 publications

(42 citation statements)

References 42 publications

(38 reference statements)

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“…It is tempting to speculate whether insertion of a charged amino acid into this loop (e.g. F241R) would reduce the capability of PMII to act as a "denaturase" (29) of hemoglobin.…”

Section: Discussionmentioning

confidence: 99%

X-ray Structure of Plasmepsin II Complexed with a Potent Achiral Inhibitor

Prade

Jones

Boss

et al. 2005

Journal of Biological Chemistry

123

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The malaria parasite Plasmodium falciparum degrades host cell hemoglobin inside an acidic food vacuole during the blood stage of the infectious cycle. A number of aspartic proteinases called plasmepsins (PMs) have been identified to play important roles in this degradation process and therefore generated significant interest as new antimalarial targets. Several x-ray structures of PMII have been described previously, but thus far, structure-guided drug design has been hampered by the fact that only inhibitors comprising a statine moiety or derivatives thereof have been published. Our drug discovery efforts to find innovative, cheap, and easily synthesized inhibitors against aspartic proteinases yielded some highly potent non-peptidic achiral inhibitors. A highly resolved (1.6 Å) x-ray structure of PMII is presented, featuring a potent achiral inhibitor in an unprecedented orientation, contacting the catalytic aspartates indirectly via the "catalytic" water. Major side chain rearrangements in the active site occur, which open up a new pocket and allow a new binding mode of the inhibitor. Moreover, a second inhibitor molecule could be located unambiguously in the active site of PMII. These newly obtained structural insights will further guide our attempts to improve compound properties eventually leading to the identification of molecules suitable as antimalarial drugs.Malaria is a major public health issue in many areas of the world, with Plasmodium falciparum being the causative agent of the most severe and deadliest form of this disease. Each year, 500 million new infections resulting in up to 2 million deaths and enormous economic damage (1) are attributed to this parasite. Drug resistance in P. falciparum has been aggravating the problem in many parts of the world during the last two decades, and new antimalarial agents addressing new targets are desperately needed.The protozoan parasite resides in erythrocytes of infected individuals during the asexual part of its life cycle. Recent studies indicated that hemoglobin degradation in a parasitic acidic organelle represents a major metabolic pathway and is crucial for survival of the parasite. Multiple proteinases appear to be actively involved in hemoglobin degradation (2-5). In particular, three members of a family of P. falciparum aspartic proteinases (PMI, 1 PMII, and PMIV) have been localized in the food vacuole (4, 5) and shown to be able to degrade hemoglobin in vitro. Another sequence-related proteinase with a new catalytic apparatus called PMIII or histo-aspartic proteinase (6) is also involved in hemoglobin catabolism in vitro. A number of research groups have reported attempts to find potent inhibitors of plasmepsins (7-12). Many of the identified molecules are peptidomimetic in nature, a compound class often associated with relatively low bioavailability and, importantly for use in developing countries, unfeasible due to significant cost of goods. We have discovered and subsequently optimized a new class of potent PMII inhibitors that could potential...

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“…It is tempting to speculate whether insertion of a charged amino acid into this loop (e.g. F241R) would reduce the capability of PMII to act as a "denaturase" (29) of hemoglobin.…”

Section: Discussionmentioning

confidence: 99%

X-ray Structure of Plasmepsin II Complexed with a Potent Achiral Inhibitor

Prade

Jones

Boss

et al. 2005

Journal of Biological Chemistry

123

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“…The protease was expressed as the mature form, lacking the pro-domain. Recombinant PM II has been shown to have activity comparable with that of native enzyme (27,28). Expression, purification, and refolding were accomplished according to the procedure described in Ref.…”

Section: Methodsmentioning

confidence: 99%

“…A 96-well fluorescence plate reader adapted to a Cary Eclipse fluorometer (Varian, Palo Alto, CA) was used. The fluorogenic peptide substrate 2837b, (EDANS-CO-CH 2 -CH 2 -CO-ALERMFLSFP-Dap-(DABCYL)OH) (AnaSpec, San Jose, CA) (28), was used at 1 M. The excitation wavelength was 336 nm, and the emission wavelength was 490 nm. Both the excitation and emission slit widths were 10 nm.…”

Section: Methodsmentioning

confidence: 99%

“…Inhibitor Competition Assay-The enzyme activity assay was modified from the published procedure (28), and the competition assay was based on the work of Tang and co-workers (29). All reactions (in triplicate) were incubated in a 96-well plate at 37°C, and the fluorescence was read at the time points of 0, 10, 60, 120, and 180 min.…”

Section: Methodsmentioning

confidence: 99%

“…Expression, purification, and refolding were accomplished according to the procedure described in Ref. 28. Mutant constructs were prepared using QuikChange (Stratagene, La Jolla, CA) with the following primers (complementary primers are not listed): 5Ј-CCATTTA-CATTTATTCTTTCTACAGGATCTGCTAATTTATGGGT-CCC-3Ј (for D34S), 5Ј-GCAAACTGTATTGTATCTAGTGGT-ACTAGTGCCATTACTGTACC-3Ј (for D214S), 5Ј-CCATTT-ACATTTATTCTTGCTACAGGATCTGCTAATTTATGGG-TCCC-3Ј (for D34A), and 5Ј-GCAAACTGTATTGTAGCTAG-TGGTACTAGTGCCATTACTGTACC-3Ј (for D214A).…”

Section: Methodsmentioning

confidence: 99%

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Hemoglobin-degrading Plasmepsin II Is Active as a Monomer

Liu¹,

Istvan²,

Goldberg

2006

Journal of Biological Chemistry

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A family of aspartic proteases called plasmepsins is important for hemoglobin degradation in intraerythrocytic Plasmodium parasites. Plasmepsin II (PM II) is the best studied member of this family. PM II and its close orthologs and paralogs form homodimers with extensive interfaces in all known crystal structures. This raised the question whether the homodimer is the functional subunit of plasmepsins in solution. We have used gel filtration chromatography, site-directed mutagenesis, and analytical ultracentrifugation to study the oligomeric status of PM II in solution. Our results reveal that PM II exists mainly as a monomer in solution and that the monomer is fully functional for catalysis. A hydrophobic loop at the PM II monomer surface, which would be buried in a PM II dimer, is shown to be essential for the hemoglobin degradation capability of PM II.

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